Synthetic excitatory amino acids

ABSTRACT

The present invention provides novel compounds that affect certain excitatory amino acid receptors, and are useful in the treatment of neurological disorders and psychiatric disorders.

This application is a continuation-in-part of Application Ser. No.08/337,349, now abandoned, filed Nov. 10, 1994, which is acontinuation-in-part of Application Ser. No. 08/289,957, filed Aug. 12,1994, now abandoned.

BACKGROUND OF THE INVENTION

In the mammalian central nervous system (CNS), the transmission of nerveimpulses is controlled by the interaction between a neurotransmitter,that is released by a sending neuron, and a surface receptor on areceiving neuron, causing excitation of this receiving neuron.L-Glutamate, which is the most abundant neurotransmitter in the CNS,mediates the major excitatory pathway in mammals, and is referred to asan excitatory amino acid (EAA). The receptors that respond to glutamateare called excitatory amino acid receptors (EAA receptors). See Watkins& Evans, Ann. Rev. Pharmacol. Toxicol., 21, 165 (1981); Monaghan,Bridges, and Cotman, Ann. Rev. Pharmacol. Toxicol., 29, 365 (1989);Watkins, Krogsgaard-Larsen, and Honore, Trans. Pharm. Sci., 11, 25(1990). The excitatory amino acids are of great physiologicalimportance, playing a role in a variety of physiological processes, suchas long-term potentiation (learning and memory), the development ofsynaptic plasticity, motor control, respiration, cardiovascularregulation, emotional states and sensory perception.

The excessive or inappropriate stimulation of excitatory amino acidreceptors leads to neuronal cell damage or loss by way of a mechanismknown as excitotoxicity. This process has been suggested to mediateneuronal degeneration in a variety of conditions. The medicalconsequences of such neuronal degeneration makes the abatement of thesedegenerative neurological processes an important therapeutic goal.

Excitatory amino acid receptors are classified into two general types.Receptors that are directly coupled to the opening of cation channels inthe cell membrane of the neurons are termed "ionotropic." This type ofreceptor has been subdivided into at least three subtypes, which aredefined by the depolarizing actions of the selective agonistsN-methyl-D-aspartate (NMDA),α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainicacid (KA). The second general type of receptor is the G-protein orsecond messenger-linked "metabotropic" excitatory amino acid receptor.This second type is coupled to multiple second messenger systems thatlead to enhanced phosphoinositide hydrolysis, activation ofphospholipase D, increases or decreases in cAMP formation, and changesin ion channel function. Schoepp and Conn, Trends in Pharmacol. Sci.,14, 13 (1993). Both types of receptors appear not only to mediate normalsynaptic transmission along excitatory pathways, but also participate inthe modification of synaptic connections during development andthroughout life. Schoepp, Bockaert, and Sladeczek, Trends in Pharmacol.Sci., 11, 508 (1990); McDonald and Johnson, Brain Research Reviews, 15,41 (1990).

The metabotropic glutamate receptors are a highly heterogeneous familyof glutamate receptors that are linked to multiple second-messengerpathways. Generally, these receptors function to modulate thepresynaptic release of glutamate, and the postsynaptic sensitivity ofthe neuronal cell to glutamate excitation. The metabotropic glutamatereceptors (mGluR) have been pharmacologically divided into two subtypes.One group of receptors is positively coupled to phospholipase C, whichcauses hydrolysis of cellular phosphoinositides (PI). This first groupare termed PI-linked metabotropic glutamate receptors. The second groupof receptors is negatively coupled to adenyl cyclase, which prevents theforskolin-stimulated accumulation of cyclic adenosine monophosphate(cAMP). Schoepp and Conn, Trends Pharmacol. Sci., 14, 13 (1993).Receptors within this second group are termed cAMP-linked metabotropicglutamate receptors. Agonists of the cAMP-linked metabotropic glutamatereceptors should be useful for the treatment of acute and chronicneurological conditions and psychiatric conditions.

Compounds have recently been discovered that affect metabotropicglutamate receptors, but have no effect on ionotropic glutamatereceptors. (1S,3R)-1-Aminocyclo-pentane-1,3-dicarboxylic acid(1S,3R-ACPD) is an agonist of PI-linked and cAMP-linked metabotropicglutamate receptors. Schoepp, Johnson, True, and Monn, Eur. J.Pharmacol., 207, 351 (1991); Schoepp, Johnson, and Monn, J. Neurochem.,58, 1184 (1992). (2S,3S,4S)-2-(carboxycyclopropyl)glycine (L-CCG-I) wasrecently described as a selective cAMP-linked metabotropic glutamatereceptor agonist; however, at higher concentrations, this compound hasactivity at PI-linked metabotropic receptors. Nakagawa, et al., Eur. J.Pharmacol., 184, 205 (1990); Hayashi, et al., Br. J. Pharmacol., 107,539 (1992); Schoepp et al., J. Neurochem., 63., page 769-772 (1994).

SUMMARY OF THE INVENTION

The present invention provides compounds that selectively affect thenegatively-coupled cAMP-linked metabotropic glutamate receptors. Morespecifically, the present invention relates to compounds of the formula##STR1## wherein: X is (CH2)_(n) ;

R² is CO₂ R⁴ and R³ is hydrogen, or R² is hydrogen and R³ is CO₂ R⁴ ;

R¹ and R⁴ are independently hydrogen, C₁ -C₁₀ alkyl, C₂ -C₁₀ alkenyl,aryl, or arylalkyl; and

n is 1;

or a pharmaceutically-acceptable salt thereof.

The present invention also provides pharmaceutical formulationscomprising a compound of formula I in combination with one or morepharmaceutically-acceptable carriers, diluents, or excipients.

Further aspects of the present invention include a method for affectingthe cAMP-linked metabotropic glutamate receptors, as well as methods fortreating a neurological disorder or a psychiatric disorder that has beenlinked to the excitatory amino acid receptors, which comprisesadministering a compound of formula I. Examples of neurologicaldisorders that are treated with a formula I compound include cerebraldeficits subsequent to cardiac bypass surgery and grafting, cerebralischemia (e.g. stroke and cardiac arrest); spinal cord trauma; headtrauma; Alzheimer's Disease; Huntington's Chorea; amyotrophic lateralsclerosis; AIDS-induced dementia; muscular spasms; migraine headaches;urinary incontinence; convulsions; perinatal hypoxia; hypoglycemicneuronal damage; drug tolerance, withdrawal, and cessation (i.e.opiates, benzodiazepines, nicotine, cocaine, or ethanol); smokingcessation; ocular damage and retinopathy; cognitive disorders;idiopathic and drug-induced Parkinson's Disease; emesis; brain edema;chronic pain; sleep disorders; Tourette's syndrome; attention deficitdisorder; and tardive dyskinesia. Examples of psychiatric disorders thatare treated with a formula I compound include schizophrenia, anxiety andrelated disorders (e.g. panic attack and stress-related disorders),depression, bipolar disorders, psychosis, and obsessive compulsivedisorders.

The present invention also provides compounds that are useful for thesynthesis of the formula I compounds. Specifically, the presentinvention relates to compounds of the formula ##STR2## wherein: X is(CH₂)_(n) ;

n is 1;

R^(2a) is CO₂ R^(4a) and R^(3a) is hydrogen, or R^(2a) is hydrogen andR^(3a) is CO₂ R^(4a) ; and

R^(4a) is hydrogen or a carboxy protecting group; and salts thereof.

The present invention also provides a process for producing a compoundof formula I, or a pharmaceutically salt thereof, which comprises:

(1) hydrolyzing a compound of the formula ##STR3## wherein: X is(CH₂)_(n) ;

n is 1;

R^(2a) is CO₂ R^(4a) and R^(3a) is hydrogen, or R^(2a) is hydrogen andR^(3a) is CO₂ R^(4a) ; and

R^(4a) is hydrogen or a carboxy protecting group;

(2) reacting a compound of the formula ##STR4## wherein X, R^(2a), andR^(3a) are as defined above, with an alkali metal cyanide and anammonium salt, and hydrolyzing the resulting intermediate as in (1)above; or

(3) hydrolyzing a compound of the formula ##STR5## wherein R^(1a) is acarboxy protecting group and X, R^(2a), and R^(3a) are as defined above;and

(4) optionally removing the carboxy protecting group; and

(5) optionally esterifying one or both carboxyl groups; and

(6) optionally separating the diastereomers and/or resolving theenantiomers; and

(7) optionally preparing a pharmaceutically-acceptable salt of theformula I compound.

DETAILED DESCRIPTION OF THE INVENTION

The term "C₁ -C₁₀ alkyl" represents a straight, branched, or cyclicalkyl chain having from one to ten carbon atoms. Typical straight orbranched C₁ -C₁₀ alkyl groups include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, heptyl,n-octyl, 2,2-dimethylhexyl, 2,5-dimethylhexyl, 2-methylheptyl,4-methylheptyl, 2,2,4-trimethylpentyl, 2,3,4-trimethylpentyl, nonyl,3,5,5-trimethylhexyl, decyl, 3,7-dimethyloctyl, and the like. The term"C₁ -C₁₀ alkyl" includes within it the terms "C₁ -C₁₆ alkyl" and "C₁ -C₄alkyl". Typical cyclic alkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and the like. Typical C₁ -C₆ alkylgroups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl.

The term "C₂ -C₁₀ alkenyl" represents straight or branched unsaturatedalkyl chains having from two to ten carbon atoms, and having one or morecarbon-carbon double bond, such as, dienes and trienes. This group alsoincludes both E and Z isomers. Representative radicals for this groupinclude vinyl, allyl, allenyl, 1-butenyl, 2-butenyl,2-methyl-1-propenyl, 3-butenyl, 2-methyl-2-propenyl, butadienyl,1-pentenyl, 2-pentenyl, 2-methyl-2-butenyl, 4-pentenyl,3-methyl-2-butenyl, 3-methyl-1,2-butadienyl, 3-hexenyl, 2-hexenyl,4-methyl-3-pentenyl, 4-hexenyl, 5-hexenyl, 3-methyl-1-penten-3-yl,4-methyl-3-pentenyl, 6-methyl-5-heptene-2-yl, 7-octenyl, 1-octen-3-yl,3-nonenyl, 2,4-dimethyl-2,6-heptadienyl, 3,7-dimethyl-6-octenyl,5-decenyl, 9-decenyl, 2,6-dimethyl-7-octenyl, and the like. The term "C₂-C₁₀ alkenyl" includes within it the term "C₂ -C₆ alkenyl".

The phrase "stereoisomeric compound" represents an optical isomer of aFormula I compound. Representative stereoisomeric compounds include the1S,2S,5R,6S isomer, 1R,2R,5S,6R isomer, the 1S,2R,5R,6S isomer, the1R,2S,5S,6R isomer, the 1S,2S,5R,6R isomer, the 1R,2R,5S,6S isomer, the1S,2R,5R,6R isomer, and the 1R,2S,5S,6S isomer.

The phrase "diastereomeric compound" represents a mixture of twonon-superimposable stereoisomers of a Formula I compound. Representativediastereomeric compounds include the 1SR,2SR,5RS,6SR mixture, the1SR,2RS,5RS,6SR mixture, the 1SR,2SR,5RS,6RS mixture, and the1SR,2RS,5RS,6RS mixture. The preferred diastereomeric compound is the1SR,2SR,5RS,6SR mixture. The preferred enantiomer is 1S, 2S, 5R, 6S.

The term "carboxy protecting group" as used herein refers to one of theester derivatives of the carboxylic acid group commonly employed toblock or protect the carboxylic acid group while reactions are carriedout on other functional groups. The protection of carboxylic acid groupsis generally described in McOmie, Protecting Groups in OrganicChemistry, Plenum Press, NY, 1973; and Greene and Wuts, ProtectingGroups in Organic Synthesis, 2nd. Ed., John Wiley & Sons, NY, 1991.Examples of such carboxy protecting groups include methyl, ethyl,methoxymethyl, methylthiomethyl, triphenylmethyl, benzyl, 4-nitrobenzyl,4-methoxybenzyl, 3,4-dimethoxy-benzyl, 2,4-dimethoxybenzyl,2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, benzhydryl, t-butyl,t-amyl, trityl, trimethylsilyl, t-butyldimethyl-silyl, allyl,1-(trimethylsilylmethyl)-prop-1-en-3-yl, and the like. Particularlypreferred carboxy protecting groups are (C₁ -C₆)alkyl groups such asmethyl and ethyl. The term "protected carboxy" refers to a carboxylicacid group having a carboxy protecting group.

The term "nitrogen protecting group" as used herein refers tosubstituents on amino groups that are commonly employed to block orprotect the amino functionality while reactions are carried out in otherfunctional groups. The protection of amino groups is generally describedin McOmie, Protecting Groups in Organic Chemistry; Plenum Press, NY,1973, and Greene and Wuts, Protecting Groups in Organic Synthesis, 2nd.Ed., John Wiley & Sons, NY, 1991. Examples of nitrogen protecting groupsinclude benzyl, t-butyl, allyl, triphenylmethyl, t-butyldimethylsilyl,triphenylsilyl, formyl, trityl, phthalimido, trichloroacetyl,chloroacetyl, phthaloyl, 2-nitrophenoxyacetyl, benzyloxycarbonyl,methoxycarbonyl, 2-methylbenzyloxycarbonyl, t-butoxycarbonyl,allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, and the like. The term"protected amino" refers to a primary or secondary amine having anitrogen protecting group.

The term "C₁ -C₄ alkoxy" represents groups such as methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, sec-butoxy, and t-butoxy. The term"halogen" refers to fluoro, chloro, bromo, and iodo.

The term "alkoxycarbonyl", means a carboxyl group having a C₁ -C₆ alkylgroup attached to the carbonyl carbon through an oxygen atom.Representatives of this group include methoxycarbonyl, ethoxycarbonyl,n-propoxycarbonyl, n-butoxycarbonyl, t-butoxycarbonyl, and the like. Thepreferred alkoxycarbonyl group is methoxycarbonyl.

The term "substituted phenyl," as used herein, represents a phenyl groupsubstituted with one or two moieties chosen from the group consisting ofhalogen, hydroxy, cyano, nitro, C₁ -C₆ alkyl, C₁ -C₄ alkoxy,alkoxycarbonyl, protected carboxy, carboxymethyl, hydroxymethyl, amino,protected amino, aminomethyl, or trifluoromethyl, Examples of asubstituted phenyl group include 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl,4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl,2-fluorophenyl, 4-hydroxyphenyl, 3-hydroxy-phenyl, 2,4-dihydroxyphenyl,3-nitrophenyl, 4-nitrophenyl, 4-cyanophenyl, 4-methylphenyl,4-ethylphenyl, 4-ethoxy-phenyl, 4-carboxyphenyl,4-(hydroxymethyl)phenyl, 4-aminophenyl, 4-propylphenyl, 4-butylphenyl,4-t-butyl-phenyl, 3-fluoro-2-methylphenyl, 2,3-difluorophenyl,2,6-difluorophenyl, 2,6-dimethylphenyl, 2-fluoro-5-methyl-phenyl,2,4,6-trifluorophenyl, 2-trifluoromethylphenyl,2-chloro-5-trifluoromethylphenyl, 2,4-bis(trifluoromethyl)-phenyl,3,5-bis(trifluoromethyl)phenyl, 2-methoxyphenyl, 3-methoxyphenyl,3,5-dimethoxyphenyl, 4-hydroxy-3-methyl-phenyl,3,5-dimethyl-4-hydroxyphenyl, 4-hydroxy-3-(hydroxymethyl)phenyl,2-amino-5-methylphenyl, 4-amino-3-trifluoromethylphenyl,3-amino-4-hydroxyphenyl, 2-methyl-4-nitrophenyl,4-methoxy-2-nitrophenyl, 2,4-dinitrophenyl, 3-cyano-4-nitrophenyl, andthe like.

The term "aryl" represents groups such as phenyl, substituted phenyl,and naphthyl. The term "arylalkyl" represents a C₁ -C₄ alkyl groupbearing one or more aryl groups. Representatives of this latter groupinclude benzyl, 2-nitrobenzyl, 4-nitrobenzyl, 1-phenylethyl,2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl, 2-methyl-2-phenylpropyl,(4-chlorophenyl)methyl, (2,6-dichlorophenyl)-methyl,bis(2,6-dichlorophenyl)methyl, (4-hydroxyphenyl)-methyl,(2,4-dinitrophenyl)methyl, triphenylmethyl,(4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)methyl,∝naphthyldiphenylmethyl, bis(2-nitrophenyl)methyl, and the like.

The term "affecting" refers to a formula I compound acting as an agonistat an excitatory amino acid receptor. The term "excitatory amino acidreceptor" refers to a metabotropic glutamate receptor, a receptor thatis coupled to cellular effectors via GTP-binding proteins. The term"cAMP-linked metabotropic glutamate receptor" refers to a metabotropicreceptor that is coupled to inhibition of adenylate cyclase activity.

The term "neurological disorder" refers to both acute and chronicneurodegenerative conditions, including cerebral deficits subsequent tocardiac bypass surgery and grafting, cerebral ischemia (for examplestroke resulting from cardiac arrest), spinal cord trauma, head trauma,Alzheimer's Disease, Huntington's Chorea, amyotrophic lateral sclerosis,AIDS-induced dementia, perinatal hypoxia, hypoglycemic neuronal damage,ocular damage and retinopathy, cognitive disorders, idiopathic anddrug-induced Parkinson's Disease. This term also includes otherneurological conditions that are caused by glutamate dysfunction,including muscular spasms, migraine headaches, urinary incontinence,drug tolerance, withdrawal, and cessation (i.e. opiates,benzodiazepines, nicotine, cocaine, or ethanol), smoking cessation,emesis, brain edema, chronic pain, sleep disorders, convulsions,Tourette's syndrome, attention deficit disorder, and tardive dyskinesia.

The term "psychiatric disorder" refers to both acute and chronicpsychiatric conditions, including schizophrenia, anxiety and relateddisorders (e.g. panic attack and stress-related cardiovasculardisorders), depression, bipolar disorders, psychosis, and obsessivecompulsive disorders.

The present invention includes pharmaceutically-acceptable salts of theformula I compounds. These salts can exist in conjunction with theacidic or basic portion of the molecule and can exist as acid addition,primary, secondary, tertiary, or quaternary ammonium, alkali metal, oralkaline earth metal salts. Generally, the acid addition salts areprepared by the reaction of an acid with a compound of formula I. Thealkali metal and alkaline earth metal salts are generally prepared bythe reaction of the hydroxide form of the desired metal salt with acompound of formula I, wherein R¹ and/or R⁴ is hydrogen.

Acids commonly employed to form such salts include inorganic acids suchas hydrochloric, hydrobromic, hydriodic, sulfuric, and phosphoric acid,as well as organic acids such as para-toluenesulfonic, methanesulfonic,oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic,and acetic acid, and related inorganic and organic acids. Suchpharmaceutically-acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, ammonium,monohydrogenphosphate, dihydrogenphosphate, meta-phosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caprate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, furmarate, hippurate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, a-hydroxybutyrate, glycolate, maleate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,napthalene-2-sulfonate, mandelate, magnesium, tetramethylammonium,potassium, trimethylammonium, sodium, methylammonium, calcium, and thelike salts.

The formula I compounds of the present invention possess four asymmetriccarbon atoms. The asymmetric centers are the substituted carbon atombearing the amino and carboxyl groups, the carbon atom where R² and R³are attached, and the two ring fusion carbon atoms. The asymmetriccarbons are at positions 2, 6, 1, and 5, respectively. Therefore, thecompounds of the present invention can exist as essentially pure opticalisomers, a mixture of two enantiomers (including racemic modifications),and a mixture of two diastereomers. When R² is CO₂ R⁴, and R¹, R³, andR⁴ are hydrogen, the biologically active and most preferredstereoisomer, as determined by receptor binding assays, has a positiveoptical rotation (α_(D)). The X-ray single crystal structure of thismost preferred enantiomer was solved, providing the relativestereochemical configuration as shown below: ##STR6##

The absolute stereochemical configuration of this most preferredenantiomer has been determined to be 1S, 2S, 5R, 6S. The presentinvention, therefore, includes the stereoisomeric Formula I compoundshaving this same preferred stereochemical configuration, mixtures ofenantiomers containing this preferred stereochemical configuration(including racemates), and mixtures of diastereomers containing thispreferred stereochemical configuration.

While all the formula I compounds of the present invention are believedto selectively affect the negatively-coupled cAMP-linked metabotropicglutamate receptors, certain compounds of the invention are preferredfor such use. Preferably, R² is CO₂ R⁴, R³ is hydrogen, and R¹ and R⁴are independently hydrogen, C₁ -C₆ alkyl, aryl, or arylalkyl.Representative compounds from this preferred group of formula Icompounds include 2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylic acid,dimethyl 2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylate, diethyl2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylate, dibutyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate, dihexyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate, diphenyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate dibenzyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate. Certain compounds of the presentinvention are more preferred for use in affecting the cAMP-linkedmetabotropic glutamate receptors. More preferably, R¹ and R⁴ areindependently hydrogen, C₁ -C₄ alkyl, aryl, or arylalkyl. Representativecompounds from this more preferred group of compounds include2-aminobicyclo- 3.1.0!hexane-2,6-dicarboxylic acid, dimethyl2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylate, diethyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate, dibutyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate, diphenyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate and dibenzyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate.

Most preferably, the compound of formula (I) is (+)-2-aminobicyclo3.1.0!hexane-2,6-dicarboxylic acid, a C₁ -C₄ alkyl, aralkyl or arylester thereof or a pharmaceutically acceptable salt thereof.

Certain compounds are most preferred for use in affecting thecAMP-linked metabotropic glutamate receptors. Most preferably R¹ and R⁴are independently hydrogen or C₁ -C₄ alkyl. Representative compoundsfrom this group of most preferred compounds include 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylic acid, dimethyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate, diethyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate, dibutyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate, and dipropyl 2-aminobicyclo3.1.0!hexane-2,6-dicarboxylate.

While all the formula X compounds of the present invention are believedto be useful for the synthesis of the formula I compounds, certaincompounds are preferred. Preferably, R^(2a) is CO₂ R^(4a) and R^(3a) ishydrogen. More preferably; R^(4a) is hydrogen or a C₁ -C₆ alkyl group,for example an ethyl group.

The formula I compounds of the present invention are generallysynthesized by cyclopropanation of a 2-cycloalken-1-one, formula IIcompound wherein X is as defined above for the formula I compounds. Theformula I compounds wherein R³ is hydrogen and R² is CO₂ R⁴ are preparedas shown in Scheme I. ##STR7##

Generally, the formula II compound is reacted with (protectedcarboxy)methyl dimethylsulfonium bromide to produce bicyclic compound offormula III wherein R^(4a) is a carboxy protecting group. This compoundis converted to an amino acid by a Strecker or a Bucherer-Bergs reactionfollowed by hydrolysis, and esterified to produce the formula IVcompounds as a mixture of isomers. This isomeric mixture is separated toproduce the formula V and formula VI compounds. These compounds are thenhydrolyzed to produce the formula I compounds wherein R² is CO₂ R⁴, andR¹ and R⁴ are hydrogen.

More specifically, a 2-cycloalken-1-one is reacted with (protectedcarboxy)methyl dimethylsulfonium bromide to produce bicyclicintermediate III. This cyclopropanation is conveniently carried in anorganic solvent in the presence of an amine base. Suitable solvents forthis reaction include acetonitrile, dichloromethane, benzene, toluene,and xylene; preferably acetonitrile or dichloromethane. Amine basessuitable for use in this reaction are non-nucleophilic bases such as1,8-diazabicyclo 5.4.0! undec-7-ene, pyridine, and collidine. Thepreferred amine base for use in this reaction is 1,8-diazabicyclo5.4.0!undec-7-ene. Preferably, the carboethoxymethyl dimethylsulfoniumbromide is reacted with the amine base, producing ethyl(dimethylsulfuranylidene)acetate in situ. The resulting mixture istreated with a 2-cycloalken-1-one. Examples 2-cycloaken-1-ones include2-cyclopenten-1-one, 2-cyclohexen-1-one, 2-cyclohepten-1-one, and2-cycloocten-1-one. The reaction is generally carried out at atemperature of about 25° C. to about 50° C., preferably at a temperatureof about 25° C. to about 30° C. The reaction is generally complete afterabout 18 hours to about three days.

Bicyclic intermediate III is converted to a bicyclic amino acid by aStrecker or a Bucherer-Bergs reaction followed by hydrolysis of theintermediates. Krauch and Kunz, Organic Name Reactions, 76, (1964) (seereferences contained therein). Preferably bicyclic ketone III is reactedwith an aqueous solution of potassium cyanide or sodium cyanide andammonium carbonate to produce hydantoin intermediates. This reaction istypically carried out in alcoholic solvent, such as ethanol or methanol.The reaction is typically carried out at a temperature of about 25° C.to about the reflux temperature of the solvent, preferably at about 50°C. The reaction is generally complete after a period of about 18 hours.The isomeric hydantoins may be isolated and purified as described below.Preferably, the mixture of isomeric hydantoins is hydrolyzed usingsodium hydroxide and subsequently esterified, without isolation orpurification, to a formula V or formula VI compound. This hydrolysis istypically carried out at the reflux temperature of the solvent for about15 hours to about 20 hours.

The products of the hydrolysis, a mixture of isomeric formula Icompounds wherein R¹ is hydrogen, are preferably esterified prior toseparation of the diastereomers and enantiomers. When the carboxyprotecting group is removed during the hydrolysis, a diester issubsequently prepared. A solution of the carboxylic acid or dicarboxylicacid in an alcohol, such as methanol, ethanol, i-propanol, or n-butanol,is treated with thionyl chloride and heated to reflux. Typically, asolution of the hydrolysis product is cooled to about 0° C. beforeaddition of the thionyl chloride. The esterification is generallycomplete after about 48 hours.

The diastereomeric products, formula V and formula VI compounds, areseparated using standard techniques. The preferred methods forseparation are crystallization and/or chromatography. The formula V andVI compounds may be selectively crystallized by formation of an acidaddition salt, such as the oxalate salt. This salt is prepared bytreating an ethyl acetate solution, containing a mixture of the formulaV and VI compounds, with oxalic acid and ethanol. Additional ethanol maybe added to aid in crystallization of one of the diastereomers. Thisprocedure gives a crystalline material enriched in one isomer, and afiltrate (mother liquor) enriched in the other isomer. The compounds maybe further purified using chromatography, such as silica-gelchromatography.

The formula V and VI compounds are hydrolyzed, if necessary, and thecarboxy protecting group removed to prepare the formula I compoundswherein R¹ and R⁴ are hydrogen. The compounds are typically hydrolyzedby treating a solution of the formula V or formula VI compound in anorganic solvent, such as tetrahydrofuran, with aqueous base, such assodium hydroxide. This hydrolysis is typically carried out at roomtemperature, and requires about 18 hours for completion. The carboxyprotecting groups are removed using standard synthetic methods. SeeMcOmie and Greene and Wuts.

The enantiomers of each diastereomeric pair of intermediate V and VIcompounds are resolved using standard resolution techniques. SeeJacques, Collet, and Wilen, Enantiomers, Racemates, and Resolutions.(1981). The preferred method for resolution of the enantiomers is theformation of diastereomeric salts between the racemic modifications andoptically active (chiral) resolving agents. Jacques, Collet, and WilenChapter 5. The formula V and VI compounds in which R^(4a) represents acarboxy protecting group can be resolved using acidic chiral resolvingagents. Examples of suitable acidic chiral resolving agents include(+)-camphoric acid, (+) and (-)-dibenzoyltartaric acid,diacetoneketogulonic acid, lasalocid, (+) and (-)-mandelic acid, (+) and(-)-malic acid, (+) and (-)-quinic acid, (+) and (-)-tartaric acid,(+)-di-p-toluoyl-D-tartaric acid, and (-)-di-p-toluoyl-L-tartaric acid.The preferred acidic resolving agents for resolution of the formula Vand VI compounds in which R^(4a) represents a carboxy protecting groupare (+)-di-p-toluoyl-D-tartaric acid, and (-)-di-p-toluoyl-L-tartaricacid. The formula V and VI compounds in which R^(4a) represents hydrogencan be resolved using basic chiral resolving agents. An example of abasic chiral resolving agent is (S)-1-phenylethylamine.

Alternatively, the bicyclic formula III compound can be converted to amixture of diastereomeric hydantoins as shown in Scheme II. ##STR8##

Bicyclic intermediate III, prepared as described above, is reacted witha solution of potassium cyanide or sodium cyanide and ammonium carbonateto produce diastereomeric hydantoin intermediates, formula VII andformula VIII compounds. This reaction is typically carried out in amixture of water and an alcohol, such as methanol or ethanol. Thereaction is carried out at a temperature of about 55° C. to about 60°C., and is generally complete after about 18 hours to about 4 days. Thediastereomeric products are separated using standard techniques, such ascrystallization and/or chromatography. Preferably, the formula VII andVIII compounds are separated by crystallization.

The formula VII and VIII compounds in which R^(4a) represents hydrogenmay be resolved using a basic chiral resolving agent. An example of abasic chiral resolving agent is (R)-1-phenylethylamine.

The hydantoin intermediate, the formula VII or VIII compound, isconverted to a formula I compound, wherein R¹ and R⁴ are hydrogen, byhydrolysis. The hydantoin group and the ester group are hydrolyzed usingaqueous base, such as sodium hydroxide, or aqueous acid, such ashydrochloric acid. This hydrolysis is typically carried out at atemperature of about 100° C. to about 150° C. The resulting formula Icompound is purified using ion-exchange chromatography.

The formula I compounds wherein R² is hydrogen and R³ is CO₂ R^(4a), areprepared as shown in Scheme III. ##STR9##

A 2-cycloalken-1-one is reacted with a carboxy protected(dimethylsulfuranylidene) acetate to produce isomeric bicyclicintermediates III and IV. This cyclopropanation is carried out in anorganic solvent at a temperature of about 45° C. to about 85° C.Suitable solvents include benzene, toluene, xylene, and acetonitrile.Preferably, the reaction is carried out in benzene at 50° C. Thediastereomeric products are separated using silica gel chromatography.The formula IX compound is converted to the formula I compounds usingthe procedures described above for conversion of the formula IIIcompounds.

The compounds of formula III in which X represents CH₂ may also beprepared as shown in Scheme IV. ##STR10##

A compound of formula XI is reacted with a (protected carboxy)methyldimethylsulfonium bromide to produce a compound of formula XII in whichR^(4a) is a carboxy protecting group. The reaction is convenientlyperformed according to a method analogous to that described herein forthe cyclopropanation of a compound of formula II. The resultant compoundof formula XII is then converted into a compound of formula XIII byheating, for example at a temperature in the range of from 160° to 500°C., preferably from 180° to 300° C. Heating the compound of formula XIIliberates cyclopentadiene. The procedure is conveniently performed underan inert gas, such as nitrogen, and in the presence of an inert organicsolvent, such as dichlorobenzene. The resultant compound of formula XIIIis then converted into a compound of formula III by reduction, forexample, by hydrogenation in the presence of palladium on charcoal. Thereduction is conveniently performed at a temperature in the range offrom 0° to 50° C. Suitable solvents for the reduction include alcohols,such as ethanol, ester such as ethyl acetate, aromatic hydrocarbons suchas toluene and amides such as dimethylformanide.

It will be appreciated that by using an optically active compound offormula XI as starting material, an optically active compound of formulaIII may be obtained.

The compounds of formula XIII are believed to be novel, and are providedas a further aspect of the invention.

The compound of formula XI (including the optically active forms) may beprepared according to the method described in Klunder et al.,Tetrahedron Lett., 1986, 27, 2543 and Takano et al, Synlett 1991, 636.

The formula I compounds wherein R¹ and R⁴ are C₁ -C₁₀ alkyl, C₂ -C₁₀alkenyl, aryl, or arylalkyl, are prepared from the correspondingcompounds wherein R¹ and R⁴ are hydrogen. These compounds are generallyprepared using standard synthetic methodologies. In a typical example,the formula I compound, wherein R¹ and R⁴ are hydrogen, can be reactedwith a C₁ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, aryl, or arylalkyl alcohol in thepresence of an acid to produce the corresponding ester. Typically, thisreaction is carried out with an excess of the alcohol in the presence ofa catalytic amount of concentrated sulfuric acid.

The formula I compounds wherein R¹ and R⁴ are not identical may beprepared from the diacid, R¹ and R⁴ are hydrogen, using standardsynthetic organic techniques. For example, the chemistry that has beendeveloped for selective functionalization of the carboxyl groups ofglutamic and aspartic acids is applicable. Alternatively, by choosing acarboxy protecting group on the formula X compound that is stable underthe hydrolysis condition for the hydantoin group, the carboxyl groupsmay be selectively manipulated.

The formula I compounds of the present invention are agonists of certainmetabotropic excitatory amino acid receptors. Specifically, the formulaI compounds are agonists of the negatively-coupled cAMP-linkedmetabotropic glutamate receptors. Therefore, another aspect of thepresent invention is a method of affecting an excitatory amino acidreceptor in mammals, which comprises administering to a mammal requiringmodulated excitatory amino acid neurotransmission apharmaceutically-effective amount of a compound of formula I. The term"pharmaceutically-effective amount" is used to represent an amount ofthe compound of the invention which is capable of affecting theexcitatory amino acid receptors. By affecting, a compound of theinvention is acting as an agonist. When a compound of the invention actsas an agonist, the interaction of the compound with the EAA receptormimics the response of the interaction of this receptor with its naturalligand (i.e. L-glutamate).

The particular dose of compound administered according to this inventionwill of course be determined by the particular circumstances surroundingthe case, including the compound administered, the route ofadministration, the particular condition being treated, and similarconsiderations. The compounds can be administered by a variety of routesincluding oral, rectal, transdermal, subcutaneous, intravenous,intramuscular, or intranasal routes. Alternatively, the compound may beadministered by continuous infusion. A typical daily dose will containfrom about 0.001 mg/kg to about 100 mg/kg of the active compound of thisinvention. Preferably, daily doses will be about 0.05 mg/kg to about 50mg/kg, more preferably from about 0.1 mg/kg to about 20 mg/kg.

A variety of physiological functions have been shown to be subject toinfluence by excessive or inappropriate stimulation of excitatory aminoacid transmission. The formula I compounds of the present invention arebelieved to have the ability to treat a variety of neurologicaldisorders in mammals associated with this condition, including acuteneurological disorders such as cerebral deficits subsequent to cardiacbypass surgery and grafting, cerebral ischemia (e.g. stroke and cardiacarrest), spinal cord trauma, head trauma, perinatal hypoxia, andhypoglycemic neuronal damage. The formula I compounds are believed tohave the ability to treat a variety of chronic neurological disorders,such as Alzheimer's disease, Hungington's Chorea, amyotrophic lateralsclerosis, AIDS-induced dementia, ocular damage and retinopathy,cognitive disorders, and idopathic and drug-induced Parkinson's. Thepresent invention also provides methods for treating these disorderswhich comprises administering to a patient in need thereof an effectiveamount of a compound of formula I.

The formula I compounds of the present invention are also believed tohave the ability to treat a variety of other neurological disorders inmammals that are associated with glutamate dysfunction, includingmuscular spasms; convulsions; migraine headaches; urinary incontinence;psychosis; drug tolerance, withdrawal, and cessation (i.e. opiates,benzodiazepines, nicotine, cocaine, or ethanol); smoking cessation;anxiety and related disorders (e.g. panic attack and stress-relateddisorders); emesis; brain edema; chronic pain; sleep disorders;Tourette's syndrome; attention deficit disorder; and tardive dyskinesia.Therefore, the present invention also provides methods for treatingthese disorders which comprise administering to a patient in needthereof an effective amount of the compound of formula I.

The compounds of the present invention are agonists of cAMP-linkedmetabotropic glutamate receptors. These compounds are negatively coupledthrough the receptor to adenyl cyclase, inhibiting the formation ofcyclic adenosine monophosphate. The formula I compounds of the presentinvention are, therefore, believed to have the ability to treat avariety of psychiatric disorders, such as schizophrenia, anxiety andrelated disorders (e.g. panic attack and stress-related disorders),depression, bipolar disorders, psychosis, and obsessive compulsivedisorders. The present invention also provides methods for treatingthese disorders which comprises administering to a patient in needthereof an effective amount of a compound of formula I.

Experiments were performed to demonstrate the ability of the formula Icompounds to affect the excitatory amino acid receptors. The affinity ofthe compounds for metabotropic glutamate receptors was demonstrated bythe selective displacement of(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive ³H!glutamate binding to rat brain cell membranes. The binding of ³H!glutamate ( ³ H!Glu) was conducted with crude membranes of ratforebrain as described by Schoepp and True. Schoepp and True,Neuroscience Lett., 145, 100-104 (1992); Wright, McDonald, and Schoepp,J. Neurochem., 63, 938-945 (1994). The concentration of the formula Icompound that inhibited 50% binding (IC₅₀), or the percent displacementof ³ H!Glu at a 10 μM or 100 μM concentration of the formula I compound,is shown in Table I.

                  TABLE I                                                         ______________________________________                                        Receptor Binding of Formula I Compounds                                       Compound.sup.a                                                                no.           IC.sub.50 (μM)                                               ______________________________________                                        3.sup.b       0.32                                                            6.sup.c       0.18                                                            7.sup.c       160.78                                                          8.sup.b       3.2                                                             ______________________________________                                         .sup.a Compound numbers are from experimental section                         .sup.b Compound tested as a mixture of enantiomers                            .sup.c Compound tested as pure enantiomer                                     .sup.d Compound tested as a mixture of diastereomers                     

Compounds 3, 6, 7 and 8 are all dicarboxylic acids. In general, it hasbeen found that the ester derivatives (those compounds of formula I inwhich one or both of R¹ and R⁴ are not hydrogen) are inactive in thereceptor binding test. However, it is believed that these compounds arehydrolysable in vivo to the corresponding acid and can thereforefunction as pro-drugs. It will be appreciated that the present inventionprovides the active dicarboxylic acids as well as any pro-drug formsthat are capable of generating the active acid in vivo.

The formula I compounds are effective in affecting the cAMP-linkedmetabotropic glutamate receptors. Representative compounds were testedfor their ability to decrease forskolin-stimulated cAMP formation in therat hippocampus and the rat cerebral cortex, using the proceduresdescribed in Schoepp and Johnson. Schoepp and Johnson, Neurochem. Int.,22, 277-283 (1993). The results of these experiments are shown in TableII.

                  TABLE II                                                        ______________________________________                                        Inhibition of Forskolin-Stimulated                                            cAMP Formation                                                                                EC.sub.50 (μM)                                             Compound no.    6         7                                                   ______________________________________                                        Rat cerebral cortex                                                                           .055 ± .017                                                                          22.0 ± 3.4                                       Rat hippocampus .036 ± .015                                                                          29.4 ± 3.04                                      ______________________________________                                    

The ability of formula I compounds to treat anxiety or a relateddisorder may be demonstrated using the well known fear potentiatedstartle and elevated plus maze models of anxiety described respectivelyin Davis, Psychopharmacology, 62:1;1979 and Lister, Psychopharmacol,92:180-185; 1987

In the fear potentiated startle model, animals are exposed to a neutralstimulus such as light (conditioned stimulus) with an aversive stimulussuch as a shock (unconditioned stimulus). Following conditioning, whenthe animals are presented with a loud acoustic stimulus, larger startleresponses are elicited when the startle stimulus is preceded by light.

The elevated plus maze model is based upon the natural aversion ofrodents to height and open spaces.

Diazepam and buspirone hydrochloride, which are clinically provenanxiolytics, are effective at reducing the fear (increased startleresponse) associated with the presentation of light in the fearpotentiated startle model, and in reducing the fear of open spaces inthe elevated plus maze model.

Male Long Evans rats (180-400 g) or male NIH Swiss mice (18-35 g) wereobtained from Harlan Sprague-Dawley, Cumberland, Ind., USA andacclimated at least 3 days before testing. Animals were housed at 23±2°C.(relative humidity 30% to 70%) and given Purina Certified Rodent Chowand water ad libitum. The photoperiod was 12 hours of light and 12 hoursof dark, with dark onset at approximately 1800 hours.

Test compounds were dissolved in a vehicle of purified water andneutralized with 5N NaOH to a pH of 7-8 when applicable. Diazepam (SigmaChemical Company, St. Louis, Mo.) was suspended in purified water by thedropwise addition of Tween 80. Control animals received the respectivevehicle.

Fear Potentiated Startle

SL-LAB (San Diego Instruments, San Diego, Calif.) chambers were used forconditioning sessions and for the production and recording of startleresponses. A classical conditioning procedure was used to producepotentiation of startle responses. Briefly, on the first 2 days, ratswere placed into dark startle chambers in which shock grids wereinstalled. Following a 5-minute acclimation period, each rat received a1 mA electric shock (500 ms) preceded by a 5 second presentation oflight (15 watt) which remained on for the duration of the shock. Tenpresentations of the light and shock were given in each conditioningsession, rats were gavaged with a solution of test compound of water andstartle testing sessions were conducted. A block of 10 consecutivepresentations of acoustic startle stimuli (110 dB, non-light-paired)were presented at the beginning of the session in order to minimize theinfluences of the initial rapid phase of habituation to the stimulus.This was followed by 20 alternating trials of the noise alone or noisepreceded by the light. Excluding the initial trial block, startleresponse amplitudes for each trial type (noise-alone vs. light+noise)were averaged for each rat across the entire test session. Data arepresented as the difference between noise-alone and light+noise. Theresult are shown in Table III.

                  TABLE III                                                       ______________________________________                                        Fear Potential Startle                                                        Test Compound ED.sub.50 (mg/kg, p.o.)                                         ______________________________________                                        Compound 6    0.3                                                             Compound 7    Inactive*                                                       Diazepam      0.4                                                             ______________________________________                                         *at highest dose tested, 10 mg/kg p.o.                                   

Automated Elevated Plus Maze

Construction of the elevated plus-maze was based on a design validatedfor mice by Lister (1987). The entire maze was made of clear Plexiglas.The maze was comprised of two open arms (30×5×0.25 cm) and two enclosedarms (30×5×15 cm). The floor of each maze arm was corrugated to providetexture. The arms extended from a central platform and angled at 90degrees from each other. The maze was elevated to a height of 45 cmabove the floor and illuminated by red light. Individual infraredphotocells were mounted along each arm of the maze to monitor closed,open, or nosepoke activity. Mice were individually placed on the centralplatform of the maze and the number of closed arm, open arm, andnosepoke (poking head only into open arm from closed arm of maze) countswere recorded and used as a measure of arm entries and time spent onvarious sections of the maze over a five-minute test period.

Oral administration of compound 6 produced significant increases in openarm activity at doses of 1, 3 and 10 mg/kg. Nosepoke counts showed asignificant increase at 3 mg/kg. Closed arm activity counts were notsignificantly altered at any dose of compound 6.

The ability of formula I compounds to protect a warm blooded mammal fromthe effects of drug withdrawal or cessation may be demonstrated using anauditory startle model. In this model, animals are dosed with a drug(nicotine or diazepam), then dosing is discontinued. This cessation ofdrug dosing elicits an increased startle response to auditory stimuli.Test compounds are then administered to animals to determine whetherthey are capable of attenuating the increased startle response.

Long Evans rats (200-400 g; Harlan Sprague Dawley, Columbus, Ind.) wereindividually housed in a controlled environment on a 12 hour light-darkcycle and given free access to food (Purina Rodent Chow) and water. Ratswere anesthetized with isoflurane and Alzet osmotic pumps (AlzaCorporation) were implanted subcutaneously.

Test compound was dissolved in a vehicle of purified water andneutralized with 5N NaOH to a PH of 7-8 when applicable. Diazepam (SigmaChemical Company, St. Louis, Mo.) was suspended in a vehicle consistingof 40% PEG 300, 10% EtOH, 2% benzyl alcohol, 1% Tween 80, and 47%purified water. Nicotine (Research Biochemicals Inc., Natick, Mass.) wasdissolved in saline. Control animals received the respective vehicle.

Nicotine withdrawal

Pumps were filled to deliver nicotine (6 mg/kg/day, s.c.), diazepam (10mg/kg/day, s.c.), test compound (0,1,3,10 mg/kg, s.c.) or vehicle.Twelve days following subcutaneous implantation of pumps, rats wereanesthetized with isoflurane and the pumps were removed. Duringwithdrawal (following pump removal), the auditory startle response (peakamplitude, Vmax) of individual rats was recorded using San DiegoInstruments startle chambers (San Diego, Calif.). Startle sessionsconsisted of a 5-minute adaptation period at a background noise level of70±2 dBA immediately followed by 25 presentations of auditory stimuli(120±2 dBA noise, 50 ms duration) presented at 8-second intervals. Peakstartle amplitudes were then averaged for all 25 presentations ofstimuli for each session and all data are presented here as overallsession means. Auditory startle responding was evaluated daily onwithdrawal days 1,2,3,4 or 5. Baseline startle responding was evaluatedprior to pump removal on day 12.

Auditory startle responding was significantly increased through thefirst three days following cessation of chronic nicotine exposure whencompared to control rats receiving water. Rats given a replacement doseof nicotine at doses of 0.03 mg/kg, i.p., or higher did not display thesame heightened startle response seen for animals with no nicotinereplacement. Pretreatment with compound 6 produced a dose-dependentblockade of the withdrawal-induced increase in startle responding aswell. A significant attenuation of the heightened startle was apparentat 3 mg/kg, p.o., dose of compound 6 when compared to nicotine controls(ED₅₀ =0.7 mg/kg, i.p.).

Diazepam Withdrawal

Auditory startle responding was significantly increased through thefirst four days following cessation of chronic diazepam exposure whencompared to control rats receiving vehicle. Replacement doses of 3 and10 mg/kg, i.p., diazepam did not block the increased startle respondingand in some instances further increased reactivity indicating tolerance.Rats which received 30 mg/kg, i.p. diazepam replacement daily 60 minutesbefore evaluation of startle responding, did not show increasedreactivity following diazepam cessation on days 1 through 4 whencompared to the diazepam control. Pretreatment with Compound 6 blockedthe expected increase in startle responding which followed cessation ofdiazepam exposure. Doses of 0.1 and 0.3 mg/kg, p.o. of compound 6significantly attenuated enhanced startle when compared to controlresponding (ED₅₀ =0.1 mg/kg, p.o.).

The compounds of the present invention are preferably formulated priorto administration. Therefore, another aspect of the present invention isa pharmaceutical formulation comprising a compound of formula I incombination with one or more pharmaceutically-acceptable carriers,diluents, or excipients. The present pharmaceutical formulations areprepared by known procedures using well-known and readily availableingredients. In making the compositions of the present invention, theactive ingredient will usually be mixed with a carrier, or diluted by acarrier, or enclosed within a carrier, and may be in the form of acapsule, sachet, paper, or other container. When the carrier serves as adiluent, it may be a solid, semi-solid, or liquid material which acts asa vehicle, excipient, or medium for the active ingredient. Thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols, ointments containing, for example, up to 10% by weight ofactive compound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, dermal patch, subcutaneous implant, and sterilepackaged powders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum, acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propyl hydroxybenzoates, talc,magnesium stearate, stearic acid, and mineral oil. The formulations canadditionally include lubricating agents, wetting agents (surfactants),emulsifying and suspending agents, preserving agents, sweetening agents,or flavoring agents. Compositions of the invention may be formulated soas to provide quick, sustained, or delayed release of the activeingredient after administration to the patient by employing procedureswell known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 1 mg to about 500 mg, more preferably about5 mg to about 200 mg of the active ingredient. The term "unit dosageform" refers to a physically discrete unit suitable as unitary dosagesfor human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical carrier, diluent, or excipient. The following formulationexamples are illustrative only and are not intended to limit the scopeof the invention in any way.

    ______________________________________                                        Formulation 1                                                                 Hard gelatin capsules are prepared using the following                        ingredients:                                                                                     Quantity                                                                      (mg/capsule)                                               ______________________________________                                        2-Aminobicyclo 3.1.0!hexane-2,6-                                                                 250                                                        dicarboxylic Acid                                                             Starch, dried      200                                                        Magnesium stearate 10                                                         Total              460          mg                                            ______________________________________                                    

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

    ______________________________________                                        Formulation 2                                                                 A tablet is prepared using the ingredients below:                                                Quantity                                                                      (mg/tablet)                                                ______________________________________                                        2-Aminobicyclo 3.1.0!hexane-2,6-                                                                 250                                                        dicarboxylic Acid                                                             Cellulose, microcrystalline                                                                      400                                                        Silicon dioxide, fumed                                                                           10                                                         Stearic acid       5                                                          Total              665          mg                                            ______________________________________                                    

The components are blended and compressed to form tablets each weighing665 mg.

    ______________________________________                                        Formulation 3                                                                 An aerosol solution is prepared containing the following                      components:                                                                                     Weight %                                                    ______________________________________                                        2-Aminobicyclo 3.1.0!hexane-2,6-                                                                  0.25                                                      dicarboxylic Acid                                                             Ethanol             29.75                                                     Propellant 22       70.00                                                     (chlorodifluoromethane)                                                       Total               100.00                                                    ______________________________________                                    

The active compound is mixed with ethanol and the mixture added to aportion of the Propellant 22, cooled to -30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

    ______________________________________                                        Formulation 4                                                                 Tablets each containing 60 mg of active ingredient are                        made as follows:                                                              2-Aminobicyclo 3.1.0!hexane-2,6-                                                                   60 mg                                                    dicarboxylic Acid                                                             Starch               45 mg                                                    Microcrystalline cellulose                                                                         35 mg                                                    Polyvinylpyrrolidone 4 mg                                                     Sodium carboxymethyl starch                                                                        4.5 mg                                                   Magnesium stearate   0.5 mg                                                   Talc                 1 mg                                                     Total                150 mg                                                   ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

    ______________________________________                                        Formulation 5                                                                 Capsules each containing 80 mg of active ingredient are                       made as follows:                                                              2-Aminobicyclo 3.1.0!hexane-2,6-                                                                   80 mg                                                    dicarboxylic Acid                                                             Starch               59 mg                                                    Microcrystalline cellulose                                                                         59 mg                                                    Magnesium stearate   2 mg                                                     Total                200 mg                                                   ______________________________________                                    

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 sieve, and filled into hard gelatincapsules in 200 mg quantities.

    ______________________________________                                        Formulation 6                                                                 Suppositories each containing 225 mg of active                                ingredient may be made as follows:                                            2-Aminobicyclo 3.1.0!hexane-2,6-                                                                  225 mg                                                    dicarboxylic Acid                                                             Saturated fatty acid glycerides                                                                   2,000 mg                                                  Total               2,225 mg                                                  ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

    ______________________________________                                        Formulation 7                                                                 Suspensions each containing 50 mg of active ingredient                        per 5 ml dose are made as follows:                                            2-Aminobicyclo 3.1.0!hexane-2,6-                                                                 50          mg                                             dicarboxylic Acid                                                             Sodium carboxymethyl cellulose                                                                   50          mg                                             Syrup              1.25        ml                                             Benzoic acid solution                                                                            0.10        ml                                             Flavor                         q.v.                                           Color                          q.v.                                           Purified water to total                                                                          5           ml                                             ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

    ______________________________________                                        Formulation 8                                                                 An intravenous formulation may be prepared as follows:                        2-Aminobicyclo 3.1.0!hexane-2,6-                                                                 100          mg                                            dicarboxylic Acid                                                             Mannitol           100          mg                                            5 N Sodium hydroxide                                                                             200          μl                                         Purified water to total                                                                          5            ml                                            ______________________________________                                    

The following Examples further illustrate the compounds of the presentinvention and the methods for their synthesis. The Examples are notintended to be limiting to the scope of the invention in any respect,and should not be so construed. All experiments were run under apositive pressure of dry nitrogen or argon. All solvents and reagentswere purchased from commercial sources and used as received, unlessotherwise indicated. Dry tetrahydrofuran (THF) was obtained bydistillation from sodium or sodium benzophenone ketyl prior to use.Proton nuclear magnetic resonance (¹ H NMR) spectra were obtained on aGE QE-300 spectrometer at 300.15 MHz, a Bruker AM-500 spectrometer at500 MHz, or a Bruker AC-200P spectrometer at 200 MHz. Free atombombardment mass spectroscopy (FABMS) was performed on a VG ZAB-2SEinstrument. Field desorption mass spectroscopy (FDMS) was performedusing either a VG 70SE or a Varian MAT 731 instrument. Optical rotationswere measured with a Perkin-Elmer 241 polarimeter. Chromatographicseparation on a Waters Prep 500 LC was generally carried out using alinear gradient of the solvents indicated in the text. The reactionswere generally monitored for completion using thin layer chromatography(TLC). Thin layer chromatography was performed using E. Merck Kieselgel60 F₂₅₄ plates, 5 cm×10 cm, 0.25 mm thickness. Spots were detected usinga combination of UV and chemical detection (plates dipped in a cericammonium molybdate solution 75 g of ammonium molybdate and 4 g of cerium(IV) sulfate in 500 mL of 10% aqueous sulfuric acid! and then heated ona hot plate). Flash chromatography was performed as described by Still,et al. Still, Kahn, and Mitra, J. Org. Chem., 43, 2923 (1978). Elementalanalyses for carbon, hydrogen, and nitrogen were determined on a ControlEquipment Corporation 440 Elemental Analyzer, or were performed by theUniversidad Complutense Analytical Centre (Facultad de Farmacia, Madrid,Spain). Melting points were determined in open glass capillaries on aGallenkamp hot air bath melting point apparatus or a Buchi melting pointapparatus, and are uncorrected. The number in parenthesis after thecompound name refers to the compound number.

Preparation 1 Carboethoxymethyl Dimethylsulfonium Bromide

A solution of ethyl bromoacetate (265 g) and dimethyl sulfide (114 g) inacetone (500 mL) was stirred at room temperature. After three days, thetitle compound was isolated by filtration of the reaction mixture.Melting point 88°-90° C.

EXAMPLE 1 (1SR,5RS,6SR) Ethyl 2-Oxobicyclo 3.1.0!hexane-6-carboxylate

A suspension of carboethoxymethyl dimethylsulfonium bromide (45.5 g) intoluene (350 mL) was treated with 1,8-diazabicyclo 5.4.0!undec-7-ene(30.2 g). The resulting mixture was stirred at room temperature. Afterone hour, the reaction mixture was treated with 2-cyclopenten-1-one(19.57 g). After an additional 18 hours, the reaction mixture was addedto a 1N hydrochloric acid/sodium chloride solution. The resultingmixture was extracted with diethyl ether. The combined ether extractswere dried over magnesium sulfate, filtered, and concentrated in vacuo.The residue was purified using silica-gel chromatography, eluting with alinear gradient of 10% ethyl acetate/hexanes to 50% ethylacetate/hexanes, to give 22.81 g of the title compound. Melting point:36°-38° C.

FDMS: m/z=168 (M+)

Analysis calculated for C₉ H₁₂ O₃ : C, 64.27; H, 7.19. Found: C, 64.54;H, 7.11.

EXAMPLE 2 (1SR,2RS, 5RS,6SR) Diethyl 2-Aminobicyclo3.1.0!-hexane-2,6-dicarboxylate (1) and (1SR,2SR,5RS,6SR) Diethyl2-Aminobicyclo 3.1.0!-hexane-2,6-dicarboxylate (2)

A solution of the compound prepared as described in Example 1 (22.81 g)in ethanol (200 mL) was treated with an aqueous solution of potassiumcyanide (9.71 g) and ammonium carbonate (21.2 g) in water (200 mL). Theresulting mixture was heated to about 50° C. After about 18 hours, thereaction mixture was allowed to cool to room temperature and treatedwith sodium hydroxide (16.2 g). The resulting mixture was heated toreflux. After about 18 hours, the reaction mixture was allowed to coolto room temperature, then cooled to 0° C. The pH of the cold mixture wasadjusted to pH 1 by the addition of concentrated hydrochloric acid. Thismixture was concentrated to dryness in vacuo. The residue was dissolvedin ethanol, cooled to 0° C., and treated with thionyl chloride (80.6 g).The resulting mixture was heated to reflux. After about 48 hours, thereaction was concentrated to dryness in vacuo. The residue was treatedwith 1N sodium hydroxide, and the resulting mixture extracted withdiethyl ether. The combined ether extracts were dried over potassiumcarbonate, filtered, and concentrated in vacuo to give 24.6 g of amixture of the title compounds.

EXAMPLE 3 (1SR,2SR,5RS,6SR) Diethyl 2-Aminobicyclo3.1.0!-hexane-2,6-dicarboxylate (2)

A solution of the compounds prepared as described in Example 2 (20.71 g)in ethyl acetate (200 mL) was treated with a solution of oxalic acid(15.46 g) in ethanol (50 mL). The resulting mixture was stirred at roomtemperature. After one hour, the reaction mixture was treated withadditional ethanol (50 mL). After 18 hours, the mixture was filtered,and the filtrate was evaporated to dryness in vacuo. The residue wastreated with 1N sodium hydroxide, and the resulting mixture extractedwith diethyl ether. The combined ether extracts were washed with brine,dried over potassium carbonate, filtered, and concentrated in vacuo. Theresidue was purified by silica-gel chromatography, eluting withmethylene chloride:5% ammonium hydroxide/methanol (97:3), to give 15.41g of the title compound.

FDMS: m/z=242 (M+H).

Analysis calculated for C₁₂ H₁₉ NO₄ : C, 59.74; H, 7.94; N, 5.81. Found:C, 59.78; H, 8.13; N, 5.77.

EXAMPLE 4 (1SR,2SR,5RS,6SR) 2-Aminobicyclo 3.1.0!hexane-2,6-dicarboxylicAcid (3)

A solution of the compound prepared as described in Example 3 (3.1 g) in2N sodium hydroxide (25 mL) and tetrahydrofuran (25 mL) was stirred atroom temperature. After about 18 hours, the tetrahydrofuran was removedunder reduced pressure, and the pH of the resulting solution wasadjusted to pH 9. The title compound was purified using ion-exchangechromatography (Bio-Rad AG1-X8), eluting with 50% acetic acid/water, togive 2.12 g. Melting point: >250° C. (dec).

FDMS: m/z=186 (M+H).

Analysis calculated for C₈ H₁₁ NO₄ : C, 51.89; H, 5.99; N, 7.56. Found:C, 51.74; H, 6.15; N, 7.45.

EXAMPLE 5 (1SR,2SR,5RS,6SR) Diethyl 2-Aminobicyclo3.1.0!hexane-2,-6-dicarboxylate (2) Hydrochloride Salt

A solution of the compound prepared as described in Example 3 (2.41 g)in diethyl ether (75 mL) was stirred at room temperature as gaseoushydrochloric acid was passed over the surface of the solution until nofurther salt formation occurred. After an additional five minutes, thesalt was removed by filtration, washed with cold diethyl ether, anddried in vacuo at 60° C. for about 18 hours, to give 2.75 g of the titlecompound. Melting point: 189-191° C.

FDMS: m/z=242 (M+H).

Analysis calculated for C₁₂ H₂₀ ClNO₄ : C, 51.89; H, 7.26; N, 5.04.Found: C, 52.03; H, 7.48; N, 5.06.

EXAMPLE 6 (-)-Diethyl 2-Aminobicyclo 3.1.0!hexane-2,6-dicarboxylate (4)

A solution of the racemic mixture of compounds prepared as described inExample 3 (6.56 g) in ethyl acetate (100 mL) was treated with a solutionof (+)-di-p-toluoyl-D-tartaric acid (12.0 g) in ethyl acetate (100 mL).After standing overnight at room temperature, the crystalline solid wasremoved by filtration and dried to give 14.7 g. Additional crystallinesolid was obtained by cooling the filtrate to 0° C. The combinedcrystalline solids were dissolved in hot ethyl acetate, containingenough 2- propanol for complete dissolution. After cooling to 0° C., thecrystalline solid was isolated by filtration, to give 2.3 g of a solidhaving an enantiomeric excess of ≧95%. The freebase form was obtained bypartitioning the salt between aqueous sodium bicarbonate and ethylacetate. The organic phase was separated, dried over potassiumcarbonate, filtered, and concentrated in vacuo to give 0.77 g of thetitle compound.

FDMS: m/z=242 (M+H).

Optical rotation: α_(D) =-5.15° (c=1, EtOH).

Analysis calculated for C₁₂ H₁₉ NO₄ : C, 59.74; H, 7.94; N, 5.81. Found:C, 59.68; H, 8.13; N, 5.58.

EXAMPLE 7 (+)-Diethyl 2-Aminobicyclo 3.1.0!hexane-2,6-dicarboxylate (5)

The mother liquors from Example 6 were combined and concentrated invacuo. The acid addition salt was converted to the freebase bypartitioning between aqueous sodium bicarbonate and ethyl acetate. Theorganic phase was separated, dried over potassium carbonate, andconcentrated in vacuo to give 3.7 g of an oil. This oil was treated with(-)-di-p-toluoyl-L-tartaric acid (7.14 g) in ethyl acetate (100 mL).After standing overnight at room temperature, the crystals werecollected by filtration and dried. The crystalline solids were dissolvedin hot ethyl acetate, containing enough 2-propanol to effect completedissolution. After cooling to 0° C., the crystals were isolated byfiltration to give 2.25 g of the title compound, having an enantiomericexcess of ≧95%. The freebase form of the title compound was obtainedsubstantially as described above, to give 0.74 g.

FDMS: m/z=242 (M+H).

Optical rotation: (α_(D) =7.22° (c=1, EtOH).

Analysis calculated for C₁₂ H₁₉ NO₄ : C, 59.74; H, 7.94; N, 5.31. Found:C, 59.81; H, 7.88; N, 5.76.

EXAMPLE 8 (+)-2-Aminobicyclo 3.1.0!hexane-2,6-dicarboxylic Acid (6)

A solution of the compound prepared as described in Example 6 (0.69 g)in tetrahydrofuran (10 mL) was treated with 1N sodium hydroxide (10 mL),and the resulting mixture vigorously stirred at room temperature. Afterseveral days, the title compound was isolated by anion-exchangechromatography (Bio-Rad AG1-X8), eluting with 50% acetic acid/water, togive 0.53 g of the title compound.

FDMS: m/z=186 (M+H).

Optical rotation: α_(D) =21.32° (c=1, 1N HCl).

Analysis calculated for C₈ H₁₁ NO₄.1.25H₂ O: C, 46.26; H, 6.55; N, 6.74.Found: C, 46.68; H, 6.47; N, 6.49.

EXAMPLE 9 (-)-2-Aminobicyclo 3.1.0!hexane-2,6-dicarboxylic Acid (7)

The title compound was prepared substantially as described in Example 8from the compound whose preparation is described in Example 7 (0.59 g).After several days, the title compound was isolated by anion-exchangechromatography (Bio-Rad AG1-X8), eluting with 50% acetic acid/water, togive 0.45 g of the title compound.

FDMS: m/z=186 (M+H).

Optical rotation: α_(D) =-22.72° (c=1, 1N HCl).

Analysis calculated for C₈ H₁₁ NO₄.H₂ O: C, 47.29; H, 6.45; N, 6.89.Found: C, 47.50; H, 6.62; N, 6.31.

EXAMPLE 10 (1SR,2SR,5RS,6RS) 2-Aminobicyclo3.1.0!hexane-2,6-dicarboxylic Acid (8)

The title compound was prepared from (1SR,2SR,5RS,6RS) diethyl2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylate substantially as describedin Examples 3 and 4.

EXAMPLE 11 (+)-Diethyl-2-Aminobicyclo 3.1.0!hexane-2,6-dicarboxylate,Hydrochloride

A stream of anhydrous HCl gas was passed over the surface of a 0° C.solution of the compound of Example 6 in anhydrous diethyl ether (75 mL)until the formation of white precipitate ceased. The resultingsuspension was stirred at room temperature for 2 hours. The reactionmixture was then diluted with diethyl ether (100 mL) and the solidfiltered under vacuum. The solid was washed with Et₂ O (250 mL) anddried under vacuum at 70° C. for 4 hours affording the title compound(2.32 g, 8.4 mmol) 77%. mp=138°-140° C. FDMS=242 M⁺ +1. Anal. calcd. forC₁₂ H₂₀ NClO₄ : C,51.89;H,7.26;N,5.04. Found C,51.61;H,7.32;N,4.99.α!_(D) =+35.52.(c=0.09,H₂ O).

EXAMPLE 12 (+)- 1R-(1a,1aα,1bβ,2b,5a,5aβ,6aα)!-1,1a,1b,2,5,5a,6,6a-octahydro-6-2,5-methanocycloprop a!indene-1-carboxylicacid, ethyl ester

A slurry of carbethoxymethyl dimethylsulfonium bromide (8.46 g, 36.9mmol) in 27 ml of acetonitrile at ambient temperature under nitrogen wastreated with 1,8-diazabicyclo 5.4.0!undec-7-ene (5.52 ml, 36.9 mmol).After stirring for 1 h the resulting yellow mixture was treated with(3aR)-3aα,4,7,7aα-tetrahydro-4α,7α-methano-1H-inden-1-one (3.60 g, 24.6mmol) as a solid in portions over 3 min. The brown reaction was allowedto stir at ambient temperature for 15 h. The reaction was quenched with5% HCl (13 ml), diluted with brine (50 ml), and washed with methylt-butyl ether (3×50 ml). The combined organic extracts were dried(MgSO₄), filtered, and concentrated in vacuo to a brown oil (5.98 g).Chromatography (100 g of flash silica gel, 8:1 then 2:1/hexanes:ethylacetate) of the crude oil provided 5.0 g (88%) of the title compound asa colorless oil determined to be a single diastereomer by HPLC analysis:α!_(D) ²⁵ +112° (c 1.39, MeOH); Rf 0.55 (hexanes:ethyl acetate/2:1); IR(CHCl₃) 2982 (w), 2938 (w), 1720 (s), 1276 (m), 1185 (m), 1048 (w) cm⁻¹; ¹ H NMR (CDCl₃) δ6.18 (dd, 1H, J=5.6, 2.9 Hz), 6.07 (dd, 1H, J=5.6,2.9 Hz), 4.14 (q, 2H, J=7.1 Hz), 3.24 (br s, 1H), 3.13 (br s, 1H), 2.86(dd, 1H, J=6.9, 4.1 Hz), 2.64 (dd, 1H, J=6.9, 5.1 Hz), 2.21-2.16 (m,2H), 1.88 (t, 1H, J=3.0 Hz), 1.57 and 1.37 (AB quartet, 2H, J=8.5 Hz),1.26 (t, 3H, J=7.1 Hz); ¹³ C NMR (CDCl₃) δ213.31, 170.78, 135.59,134.16, 61.56, 51.47, 51.17, 46.45 (2 carbons), 44.20, 39.76, 32.75,25.76, 14.56. Anal. calcd. for C₁₄ H₁₆ O₃ : C, 72.39; H, 6.94. Found: C,72.63; H, 7.08.

EXAMPLE 13 (+)- 1(R),5(S),6(R)!-Bicyclo3.1.0!hex-3-en-2-one-6-carboxylic acid ethyl ester.

A solution of the product of Example 12 (4.89 g, 21.1 mmol) in 14 ml ofdry dimethyl sulfoxide was heated to reflux with simultaneous stirringand purging with nitrogen (sub-surface needle) to drive off liberatedcyclopentadiene for 24 h. The reaction was cooled to room temperature,diluted with methyl t-butyl ether (100 ml), and washed with water (1×50ml). The aqueous layer was washed with methyl t-butyl ether (1×25 ml)and the combined organic extracts were dried (MgSO₄), filtered, andconcentrated in vacuo to a light yellow solid. The crude cyclopentenonewas crystallized from hexanes-methyl t-butyl ether to afford 1.91 g(55%) of the title compound: mp 96°-98 ° C.; α!_(D) ²⁵ +251° (c 1.12,MeOH); Rf 0.49 (hexanes:ethyl acetate/2:1); IR (KBr) 2997 (w), 1728 (s),1747 (s), 1696 (s), 1292 (m), 1266 (s), 1190 (s), 1177 (s) cm⁻¹ ; ¹ HNMR (CDCl₃) δ7.61 (dd, 1H, J=5.6, 2.5 Hz), 5.74 (d, 1H, J=5.6 Hz), 4.15(q, 2H, J=7.1 Hz), 2.96-2.94 (m, 1H), 2.62 (br t, 1H, J=3.9 Hz), 2.26(t, 1H, J=2.8 Hz), 1.27 (t, 3H, J=7.1 Hz); ¹³ C NMR (CDCl₃) δ203.56,168.28, 159.96, 129.99, 61.70, 46.19, 30.39, 29.28, 14.49. Anal. calcd.for C₉ H₁₀ O₃ : C, 65.05; H, 6.07. Found: C, 64.78; H, 6.24.

EXAMPLE 14 (-)- 1(R),5(S),6(R)!-Bicyclo 3.1.0!hexan-2-one-6-carboxylicacid ethyl ester

A solution of the product of Example 13 (1.73 g, 10.4 mmol) in 35 ml of95% ethanol under nitrogen was treated with 10% Pd/C (87 mg, 5 wt %).The flask was purged with hydrogen and stirring was maintained under ahydrogen atmosphere (balloon pressure) for 5 h at which time another 35mg (2 wt %) of 10% Pd/C was added. The mixture was allowed to stir underhydrogen for an additional 50 min. The flask was purged with nitrogenand the catalyst was removed via filtration through celite, washing withethyl acetate. The filtrate and washings were concentrated in vacuo to ayellow solid (1.75 g). The crude solid was crystallized fromhexanes-methyl t-butyl ether to afford 1.38 g (79%) of the titlecompound: mp 63°-65° C.; α!_(D) ²⁵ -60° (c 1.34, MeOH); Rf 0.49(hexanes:ethyl acetate/2:1); IR (KBr) 2987 (w), 1722 (s), 1410 (m), 1193(s), 1009 (m), 827 (m) cm⁻¹ ; ¹ H NMR (CDCl₃) δ4.16 (q, 2H, J=7.1 Hz),2.52 (q, 1H, J=4.9 Hz), 2.29-2.22 (m, 2H), 2.17-2.00 (m, 4H), 1.28 (t,3H, J=7.1 Hz); ¹³ C NMR (CDCl₃) δ212.07, 170.80, 61.64, 36.17, 32.30,29.59, 26.91, 22.87, 14.56. Anal. calcd. for C₉ H₁₂ O₃ : C, 64.27; H,7.19. Found: C, 64.10; H, 7.31.

EXAMPLE 15 (+)- 1(R),2(R),5(S),6(R),5'(R)!-2-Spiro-5'-hydantoinbicyclo3.1.0!hexane-6-carboxylic acid ethyl ester

A mixture of the produce of Example 14 (1.20 g, 7.13 mmol), potassiumcyanide (511 mg, 7.85 mmol), and ammonium carbonate (1.37 g, 7.13 mmol)in 7.1 ml of 95% ethanol and 2.9 ml of water was allowed to stir at 36°C. for 10 h and at room temperature for 13 h. The cloudy yellow reactionwas cooled to 0° C. and diluted with 7.8 ml of cold water. Afterstirring for 1.5 h the white solid was collected and washed with coldwater (2×5 ml). The solid was dried in vacuo to provide 1.17 g (69%) ofthe title compound as a single diastereomer as determined by HPLCanalysis: mp 247°-249° C.; α!_(D) ²⁵ +23° (c 1.05, MeOH); IR (KBr) 3504(m), 3262 (m), 2983 (w), 2766 (w), 1771 (m), 1723 (s), 1415 (m), 1182(w) cm⁻¹ ; ¹ H NMR (DMSO-d₆) δ10.58 (s, 1H), 7.93 (s, 1H), 4.06 (q, 2H,J=7.1 Hz), 2.08-2.01 (m, 1H), 1.94-1.83 (m, 4H), 1.79 (dd, 1H, J=13.9,8.5 Hz), 1.40-1.33 (m, 1H), 1.20 (t, 3H, J=7.1 Hz); ¹³ C NMR (DMSO-d₆)δ178.30, 172.62, 157.01, 69.52, 61.04, 33.86, 30.37, 28.27, 26.49,20.95, 14.93. Anal. calcd. for C₁₁ H₁₄ N₂ O₄ : C, 55.46; H, 5.92; N,11.76. Found: C, 55.76; H, 5.95; N, 11.84.

EXAMPLE 16 (-)- 1(R),2(R),5(S),6(R)!-2-Aminobicyclo3.1.0!hexane-2,6-dicarboxylic acid

A solution of the product of Example 15 (976 mg, 4.10 mmol) in 8.2 ml of3N NaOH was allowed to reflux with stirring for 24 h. Upon cooling toroom temperature the reaction was applied directly to an ion exchangecolumn (prepared from 50 g of Bio-Rad AG 1-X8 acetate resin prepared viawashing with 50 ml of 1N NaOH followed by 50 ml of water followed by,after application of the reaction mixture, another 50 ml of 1N NaOH)eluting with 1:1/water:acetic acid, collecting 50 ml fractions.Fractions 2 and 3, which contained product, were combined andconcentrated in vacuo providing 770 mg of a white solid. The solid wasslurried in 4 ml of water and filtered, washing with water (1×4 ml). Thesolid was dried in vacuo at 40 ° C. to afford 634 mg (76%) of the titlecompound as a white powder: IR (KBr); 3235(br;s), 2971(m), 2016(br,w),1694(m), 1613(s), 1509(m), 1237(m) (cm-1), ¹ H NMR (trifluoroaceticacid-d) δ2.76-2.74 (m, 1H), 2.65-2.52 (m, 3H), 2.38-2.31 (m, 2H),1.96-1.88 (m, 1H); ¹³ C NMR (trifluoroacetic acid-d) δ179.43, 175.63,69.53, 34.92, 31.75, 31.66, 27.63, 23.04. An analytical sample wasprepared by crystallization from water: mp 277°-280 ° C. (dec); α!_(D)²⁵ 23° (c 1.35, 1N HCl)

EXAMPLE 17 2-Oxobicyclo 3.1.0!hexane-6-carboxylic acid

A mixture of 60 g of ethyl 2-oxobicyclo 3.1.0!hexane-6-carboxylate and300 ml of 1N sodium hydroxide was stirred at 25°-30° C. After 2.5 hours,concentrated hydrochloric acid was added to adjust the pH to 0.8-1.2.The resulting solution was extracted with ethyl acetate. The extractswere dried over magnesium sulfate, filtered, and concentrated to give49.1 g (98%) of the crude material. Recrystallization from 100 ml ofethyl acetate gave the title compound, mp 123.5°-128° C.

FDMS: m/z=140 (M+)

Analysis calculated for C₇ H₈ O₃ : C, 60.00; H, 5.75. Found: C, 60.14;H, 5.79.

EXAMPLE 18 2-Oxobicyclo 3.1.0!hexane-6-carboxylic acid salt with(S)-1-phenylethylamine

A solution of 14 g of the compound prepared in Example 17 in 140 ml of25% ethanol in ethyl acetate was combined with (S)-1-phenylethylamine (1eq.). After stirring overnight, the precipitated salt was isolated byfiltration and dried to give 11.87 g (45.4%) of the desired salt.Conversion of the salt to the partially resolved 2-oxobicyclo3.1.0!hexane-6-carboxylic acid by the method of Example 17 and analysisindicated that the salt was 68% ee. The enantiomeric excess wasdetermined by conversion to the methyl ester with diazomethane followedby chiral HPLC on a Chiralpak AS column at 40° C. eluted with 10%isopropanol/90% hexane at 1 ml/min with detection at 210 nm.

EXAMPLE 19 (+)-2-Oxobicyclo 3.1.0!hexane-6-carboxylic acid

A mixture of 1.31 g of the product of Example 18 and 10 ml of 1Nhydrochloric acid was stirred for 5 minutes and extracted with ethylacetate. The extracts were dried over sodium sulfate, filtered, andconcentrated to give 0.61 g of the title compound, mp 110°-115° C. Theproduct was determined to be 68% ee by chiral HPLC (method of Example18)

FDMS: m/z=141 (M+H)

Optical Rotation: α_(D) =49.85°

EXAMPLE 20 (-)-2-Spiro-5'-hydantoinbicyclo 3.1.0!hexane-6-carboxylicacid

A solution of the compound prepared as described in Example 19 (68% ee,1 eq.), potassium cyanide (1.25 eq.), and ammonium carbonate (2.5 eq)were combined and stirred in ethanol/water at 25° C. for 40 hours. Themixture was acidified with 6N hydrochloric acid, concentrated, dilutedwith water, and filtered to give a 79% yield of a 90:10 mixture ofdiastereomers, mp 286°-290° C. The diastereomeric mixture wasrecrystallized from isopropanol/water to give in 48% yield the titlecompound in 100% diastereomeric and 100% enantiomeric purity(enantiomeric ratio determined by chiral HPLC on a 4.6×150 mm ChiralcelOD-H column, eluted with 15 % isopropanol/85% hexane at 1 ml/min at 40°C. with detection at 220 nm; diastereomeric ration determined by HPLC ona Zorbax SB-phenyl column at 40° C. with elution with 90:10buffer/acetonitrile eluted at 2 ml/min with detection at 220 nm(buffer=0.1M dibasic sodium phosphate monohydrate adjusted to pH 2.1with phosphoric acid).

FDMS: m/z=211 (M+H)

Optical Rotation: α_(D) =-25.98°

Analysis calculated for C₉ H₁₀ N₂ O₄ : C, 51.43; H, 4.79; N, 13.33.Found: C, 51.38; H, 4.80; N, 13.26.

EXAMPLE 21 Ethyl 2-spiro-5'-hydantoinbicyclo 3.1.0!hexane-6-carboxylate

A mixture of 5.05 g of ethyl 2-oxobicyclo 3.1.0!hexane-6-carboxylate,2.15 g of potassium cyanide, 5.77 g of ammonium carbonate, 30 ml of 2B-3ethanol, and 12 ml of water was stirred at 35° C. until the reaction wascomplete by HPLC.

After 15 hours, the reaction mixture was cooled to 0° C. and 33 ml ofwater was added to the mixture. After 2 hours at 0° C., the precipitatewas isolated by filtration and dried to give 5.23 g (73%) of the titlecompound, mp 217°-220 ° C.

FDMS: m/z=238.1 (M+)

Analytical calculated for C₁₁ H₁₄ N₂ O₄ : C, 55.46; H, 5.92; N, 11.76.Found: C, 55.74; H, 5.88; N, 11.50.

EXAMPLE 22 2-Spiro-5'-hydantoinbicyclo 3.1.0!hexane-6-carboxylic acid

A mixture of 16.32 g of the product of Example 21 and 137 ml of 2N NaOHwas stirred at 25° C. After 1 hour, concentrated hydrochloric acid wasadded to adjust the pH to 1.0. The resulting precipitate was isolated byfiltration and dried to give 13.70 g (95%) of the title compound, mp277°-279° C.

FDMS: m/z=210.1 (M+)

Analysis Calculated for C₉ H₁₀ N₂ O₄ : C, 51.43; H, 4.79; N, 13.33.Found: C, 51.70; H, 4.93; N, 13.43.

EXAMPLE 23 2-Spiro-5'-hydantoinbicyclo 3.1.0!hexane-6-carboxylic acid,(S)-1-phenylethylamine salt

A mixture of 1.05 g of the product of Example 22 and 16.6 ml of a 1.6:1solution of acetone:water was stirred at 25° C. while adding 1.53 g ofR-(+)-1-phenylethylamine. The mixture was stirred for 2 hours at roomtemperature. The crystals were filtered, rinsed with acetone, and driedto give 0.74 g (45%) of the title compound, mp 205°-212°° C.

Optical Rotation: α_(D) =-31.88° (c=1, methanol)

EXAMPLE 24 (-)-2-Spiro-5'-hydantoinbicyclo 3.1.0!hexane-6-carboxylicacid

A mixture of 0.74 g of the product of Example 23 and 10 ml of water wasstirred at 25° C. while the pH was adjusted from 6.81 to 1.0 using 1NHCl. The reaction mixture was stirred for 1 hour and the product wascollected by filtration and dried to give 0.35 g (75%) of the titlecompound, mp 310° C. (decomp).

FDMS: 210.1 (M+)

Optical Rotation: α_(D) =-24.22° (c=1, methanol)

Analysis calculated for C₉ H₁₀ N₂ O₄ : C, 51.43; H, 4.80; N,13.33.Found: C, 51.67; H, 4.87; N, 13.61.

EXAMPLE 25 (+)-2-Aminobicyclo 3.1.0!hexane-2,6-dicarboxylic Acid

A solution of 184 g of (-)-2-spiro-5'-hydantoinbicyclo3.1.0!hexane-6-carboxylic acid and 1750 ml of 3N NaOH was heated atreflux until the reaction was complete by HPLC. After 28 hours, thesolution was cooled to room temperature and filtered through glass paperto remove trace amounts of insoluble material. The pH of the solutionwas adjusted to 3.0 using concentrated HCl. The reaction mixture wasstirred 1 hour at room temperature and two hours at 0° C. Theprecipitated product was collected by filtration, washed with 170 ml ofcold water and dried to give 152.5 grams (86%) of the title compound.

FDMS: m/z=186.1 (M+l)

Optical rotation: α_(D) =23.18° (c=1, 1N HCl)

We claim:
 1. A compound of the formula ##STR11## wherein: X is (CH₂)_(n) ;R² is CO₂ R⁴ and R³ is hydrogen, or R² is hydrogen and R³ is CO₂ R⁴ ; R¹ and R⁴ are independently hydrogen, C₁ -C₁₀ alkyl, C₂ -C₁₀ alkenyl, aryl, or arylalkyl; and n is 1; or a pharmaceutically-acceptable salt thereof.
 2. The compound of claim 1 wherein R² is CO₂ R⁴ and R³ is hydrogen; or a pharmaceutically-acceptable salt thereof.
 3. The compound of claim 2 wherein R¹ and R⁴ are independently hydrogen, C₁ -C₆ alkyl, aryl, or arylalkyl, or a pharmaceutically-acceptable salt thereof.
 4. The compound of claim 2, or a pharmaceutically-acceptable salt thereof, having the relative stereochemical configuration as shown below: ##STR12##
 5. The compound of claim 4 wherein R¹ and R⁴ are independently hydrogen, C₁ -C₄ alkyl, arylalkyl, or aryl, or a pharmaceutically-acceptable salt thereof.
 6. The compound of claim 5 wherein R¹ and R⁴ are independently hydrogen, or C₁ -C₄ alkyl, or a pharmaceutically-acceptable salt thereof.
 7. The diastereomeric compound of claim 5 which is (1SR,2SR,5RS,6SR) 2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylic acid, or a pharmaceutically-acceptable salt thereof.
 8. The stereoisomeric compound of claim 5, which is (+)-2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylic acid, a C₁ -C4 alkyl, aralkyl or aryl ester thereof or a pharmaceutically acceptable salt thereof.
 9. The stereoisomeric compound of claim 8, which is (+)-2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylic acid, a C₁ -C₄ alkyl ester thereof, or a pharmaceutically acceptable salt.
 10. The stereoisomeric compound of claim 9 which is (+)-2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylic acid, or a pharmaceutically-acceptable salt thereof.
 11. The stereoisomeric compound of claim 10 which is (+)-2-aminobicyclo 3.1.0!hexane-2,6-dicarboxylic acid.
 12. A pharmaceutical formulation comprising a compound of claim 1 in combination with one or more pharmaceutically-acceptable carriers, diluents, or excipients.
 13. A pharmaceutical formulation comprising the compound of claim 8 in combination with one or more pharmaceutically-acceptable carriers, diluents, or excipients. 